Editing a mushroom with high-digestibility using a novel endo-N-acetyl-β-D-glucosaminidase

Fungi comprise approximately 2 % of the Earth's biomass; however, the human gastrointestinal tract has a limited capacity to digest fungal biomass. In this study, a novel endo-N-acetyl-β-D-glucosaminidase, Endo CM, was characterized in the mushroom-forming fungus Cordyceps militaris, where it plays a role in maintaining the integrity of the fungal cell wall. Through gene editing, the Endo CM promoter was engineered to remove the binding site of the CmCreA carbon catabolite repressor, and the transformant was named CmT. After 12 h of treatment with simulated digestive fluids, the residual mycelial biomass of CmT was reduced to 50.00 ± 1.57 %, compared with 69.47 ± 0.97 % (p = 0.00005) for the parent strain. CmT also released more amino acids during the simulated digestion, suggesting that the expression level of Endo CM affects the accessibility of mycelial biomass to digestive enzymes. Additionally, CmT produced fruiting bodies with improved flavor but impaired appearance. This study highlights the production of alternative proteins with high digestibility and provides a sustainable approach for breeding mushrooms with improved digestibility and absorption properties.